Vibrio cholerae is a facultative pathogen and is the causative agent of the severe secretory diarrheal disease cholera. Virtually all cholera at present is caused by El Tor biotype strains which differ biochemically and phenotypically from strains of the better-characterized classical biotype. The goal of the proposed work is to understand the pathogenicity and physiology of El Tor V. cholerae during intestinal infection. Laboratory manipulation of virulence factor expression in El Tor strains is difficult, and this has hampered their study. Moreover, knowledge of which genes are expressed by V. cholerae during infection is rudimentary, and virtually nothing is known about the patterns of expression of such genes within the gastrointestinal tract. Advanced genetic approaches will be used to identify and analyze El Tor infection-induced genes within the suckling mouse model of cholera, and during colonization of a natural plankton host. The regulation and spatiotemporal patterns of expression of genes in the ToxR regulon, and newly identified virulence genes, will be determined in vivo using an enhanced recombinase-based in vivo expression technology (RIVET). Factors which are non-essential for in vitro growth but are essential for colonization of the small bowel of suckling mice, will be comprehensively identified using a modified Signature-Tagged Mutagenesis (STM) procedure. As a complementary approach, RIVET will be used to identify genes induced transcriptionally during infection, some of which may be essential for in vitro growth. These methods will also be used to identify and characterize V. cholerae genes important for colonizing the plankton host Anabaena variabilis. The importance of select genes for colonization of each host will be determined by constructing specific gene mutations followed by colonization studies. The broad specificity of RIVET, combined with the focused specificity of STM, will allow identification of an unprecendented number and variety of genes that play roles in the physiology and virulence of V cholerae in these animal and environmental host systems. These studies will not only establish a basis for understanding the dynamics of virulence gene expression during infection of an intact host, but will aid in the development of new cholera vaccines and suggest new approaches for the prevention of the dissemination of this lethal organism.